Duchenne muscular dystrophy (DMD) is the most frequent progressive muscle degenerative disease, affecting approximately one in 3,500 to 5000 male births. DMD is caused by deletions or mutations in the gene encoding dystrophin, located on the X chromosome. Dystrophin is required for the assembly of the dystrophin-glycoprotein complex, and provides a mechanical and functional link between the cytoskeleton of the muscle fiber and the extracellular matrix. The absence of functional dystrophin causes fiber degeneration, inflammation, necrosis and replacement of muscle with scar and fat tissue, resulting in progressive muscle weakness and premature death due to respiratory and cardiac failure between the second and fourth decade of life (Moser, H., Hum Genet, 1984. 66(1): 17-40).
A milder form of the disease called Becker muscular dystrophy (BMD) is distinguished from DMD by delayed onset, later dependence on wheelchair support, and longer life span. BMD is caused by mutations maintaining the reading frame and the most critical parts of the gene, leading to a truncated but still functional dystrophin protein (Muntoni F et al, Lancet Neurol, 2003. 2(12): 731-40).
There is no cure nor effective treatment available for DMD (Rodino-Klapac, L. R. et al., Curr Neurol Neurosci Rep, 2013. 13(3): 332) or BMD. Conventional therapies are limited to supportive care, which partially alleviates signs and symptoms, but does not directly target the disease mechanism nor reverse the phenotype.
There currently are several therapeutic strategies being developed for DMD including in vivo gene therapy, cell transplantation therapy, pharmacologic rescue of DMD nonsense mutations and exon skipping strategies to repair the dystrophin gene reading frame. All of these strategies have problems to overcome, including targeting different muscle groups, optimization of delivery, long-term expression of the transgene, and potential immune response (Jarmin et al., Expert Opin Biol Ther, 2014. 14(2): 209-30).
Different gene transfer approaches for DMD aim to compensate for dystrophin loss-of-function and offer the potential to treat all patients using a single medication. In order to prevent muscle degeneration, around 30% of normal levels of dystrophin proteins are likely to be required.
The dystrophin gene is the largest known gene in the human genome, spanning over 2.5 Mb or some 2% of the entire X chromosome in humans. It consists of 79 exons gene locus (full length cDNA: 11.1 kb), which encodes for a 3685 amino acids, 427 kD dystrophin protein. Dystrophin protein is defined by four structural regions (FIG. 1). These are the actin binding domain at the NH2 terminus (exons 1 to 8), central rod domain (24 spectrin-like repeats R1-24 and 4 Hinge regions H1-4; exons 9 to 62), cysteine-rich (CR) domain (exons 63 to 69), and carboxy-terminal (CT) domain (exons 70 to 79).
This size is too large to fit inside known gene therapy vector systems, especially in Adeno-Associated Virus (AAV) vector which is one of the promising candidates with efficient gene transfer into various muscle groups depending on tropism of AAV serotypes. AAV vector has a potential to show long term gene transduction in both dividing (myofibres and cardiomyocytes) and non-dividing (mature myotubes) muscle cells.
Indeed, a major limitation of AAV is its cargo capacity which is thought to be limited to around 5 kb, the size of parental viral genome (Wu Z. et al., Mol Ther., 2010, 18(1): 80-86; Lai Y. et al., Mol Ther., 2010, 18(1): 75-79; Wang Y. et al., Hum Gene Ther Methods, 2012, 23(4): 225-33). Larger vector genomes resulted in truncated packaged genomes, heterogeneous population of genome with broad size distribution, and lower expression efficiency (Wu Z. et al., Mol Ther., 2010, 18(1): 80-86). The use of proteasome inhibitors has been suggested to improve the transduction profile of AAV encapsidating genomes larger than wild-type size (Grieger and Samulski, J. Virol. 2005, 79(15): 9933-44).
However, packaging of a 5.4 kb DNA sequence has been reported for a cardiac sarcomeric protein produced with AAV6 or AAV9 vectors in cardiac tissue (Mearini et al., Nature Communications, 2014. 5:5515).
To overcome the DNA packaging limitation of AAV (<5 kb), several research groups have attempted to engineer synthetic microdystrophins (MD, also known as “minidystrophin”), i.e. truncated but functional proteins. A series of microdystrophins have been designed to encode truncated dystrophins, optimized to contain the more clinically important regions of the protein. Such regions have generally been thought to lie within dystrophin's N-terminal and cysteine-rich domains.
Microdystrophin, which contains the first 3 and the last of the 24 spectrin-like repeats without the C-terminal domain (ΔR4-R23/ΔCT), named MD1, displayed highly functional activity to restore dystrophin and co-localise with syntrophin and dystrobrevin, but it failed to recruit nNOS at the sarcolemma in mdx mouse model (Yue, et al., Mol Ther, 2006. 14(1): 79-87).
Recent trials with AAV2/8 vector encoding a sequence optimized canine MD1 micro-dystrophin, with expression driven by a muscle-specific spc512 promoter (AAV8-spc512-cMD1) in the dystrophic CXMDj dog (Koo et al., J Gene Med, 2011. 13(9): 497-506) have proved encouraging. Isolated limb perfusion studies in 3-month-old animals using modest single administration vector doses (5×1012/kg) demonstrate up to 95% Dystrophin positive fibres in the treated limbs at the 6-week post-treatment and significant normalisation of clinical scores in treated canine subjects.
However, the relevance of the deleted regions, especially of the CT domains of dystrophin, in muscle function remains controversial.
Therefore, there is a need in the art for developing partially deleted but highly functional dystrophin genes, which can be successfully packaged inside AAV vectors.